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1.
The modification reaction of 28 S rRNA in eukaryotic ribosomes by ricin A-chain was characterized. To examine whether ricin A-chain release any bases from 28 S rRNA, rat liver ribosomes were incubated with a catalytic amount of the toxin, and a fraction containing free bases and nucleosides was prepared from the postribosomal fraction of the reaction mixture by means of ion-exchange column chromatography. Thin-layer chromatographic analysis of this fraction revealed a release of 1 mol of adenine from 1 mol of ribosome. When the ribosomes or naked total RNAs were treated with ricin A-chain in the presence of [32P] phosphate, little incorporation of the radioactivity into 28 S rRNA was observed, indicating that the release is not mediated by phosphorolysis. Thus, considering together with the previous result (Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) J. Biol. Chem. 262, 5908-5912), the results in the present experiments demonstrated that ricin A-chain inactivates the ribosomes by cleaving the N-glycosidic bond of A4324 of 28 S rRNA in a hydrolytic fashion.  相似文献   

2.
Y Endo  K Tsurugi  H Franz 《FEBS letters》1988,231(2):378-380
The site of action of the A-chain of mistletoe lectin (ML-A) from Viscum album on eukaryotic ribosomes was studied. Treatment of rat liver ribosomes with ML-A, followed by treatment of the isolated rRNA with aniline, caused the release of a fragment with about 450 nucleotides from 28 S rRNA. Further analysis of nucleotide sequences of this fragment revealed that the aniline-sensitive site of phosphodiester bond was between positions A-4324 and G-4325 in 28 S rRNA. These results indicate that ML-A inactivates the ribosomes by cleaving a N-glycosidic bond at A-4324 of 28 S rRNA in the ribosomes as ricin A-chain does.  相似文献   

3.
The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes was studied. Treatment of ribosomes with any one of these proteins caused the 28S rRNA extracted from the inactivated ribosomes to become sensitive to treatment with aniline. A fragment containing about 450 nucleotides was released from the 28S rRNA. Further analysis of the nucleotide sequences of the 450-nucleotide fragments revealed that the aniline-sensitive phosphodiester bond was between A-4324 and G-4325 of the 28S rRNA. These results indicate that all six ribosome-inactivating proteins damage eukaryotic ribosomes by cleaving the N-glycosidic bond at A-4324 of the 28S rRNA of the ribosomes, as does ricin A-chain.  相似文献   

4.
Y Endo  T Oka  K Tsurugi  H Franz 《FEBS letters》1989,248(1-2):115-118
A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline-sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N-glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A-chain.  相似文献   

5.
In a previous report (Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130) it was shown that the RNA N-glycosidase activity of ricin A-chain was responsible for the ability of this protein to inactivate eukaryotic ribosomes. The objective of the present study was to determine whether a similar mechanism was used by a ribosome-inactivating protein from pearled barley (barley toxin). Rat liver ribosomes were incubated either with ricin A-chain or barley toxin, and the rRNA was extracted and treated with acidic aniline to hydrolyze phosphodiester bonds rendered susceptible by removal of a purine or pyrimidine base. Evaluation of the rRNA by polyacrylamide/agarose electrophoresis disclosed two 28 S rRNA-derived fragments which differed in size from those generated by untreated (control) ribosomes. Sequencing of the smaller of these fragments confirmed that - as is the case for ricin A-chain - the aniline-sensitive site in barley toxin-treated ribosomes was between A and G in 28 S rRNA. We conclude that barley toxin inactivates ribosomes via a mechanism identical to that of ricin A-chain: enzymatic hydrolysis of the N-glycosidic bond at A of 28 S rRNA.  相似文献   

6.
Gu YJ  Xia ZX 《Proteins》2000,39(1):37-46
Four substrate analogs-nicotinamide adenine dinucleotide, adenylyl (3', 5') guanosine, guanylyl (3',5') adenosine, and adenosine 2', 5'-diphosphate-have been used to prepare the complexes with trichosanthin (TCS), a type I ribosome-inactivating protein that possesses the activity of N-glycosidase. The crystal structures of the complexes have been determined and refined at high resolution. The refined structures show that the N-glycosidic bonds of all the four substrate analogues are hydrolyzed and a common structure is shared by the four complexes, in which only adenine, the product of the enzymatic reaction, is bound in the active center. The structure is compared with those of native trichosanthin and a previously reported trichosanthin-NADPH complex in which the N-glycosidic bond is uncleaved. The structural comparison shows that the conformation of Tyr70 obviously differs from those in the latter two structures, i.e., the side chain of Tyr70 is rotated along its Cbeta-Cgamma bond by approximately 70 degrees. The water molecule found to be preassociated with the N-glycosidic bond in the TCS-NADPH complex structure and proposed to be the water candidate responsible for hydrolyzing the N-glycosidic bond disappears in the trichosanthin-product complex structure. Based on the comparison of the three structures representing the different stages of the enzymatic reaction, the catalytic mechanism of RNA N-glycosidase has been further elucidated. Proteins 2000;39:37-46.  相似文献   

7.
Trichosanthin(TCS)isatypeIribosomeinactivatingprotein(RIP)[1].ThemechanismofinactiveribosomebelongstothatofRNANglycosidase[2].TheactivepocketofNglycosidasehasbeenestablishedonthesurfacevacantbetweentwodomainsthroughcrystalstructuredetermination[3]andtheacti…  相似文献   

8.
Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA(2)) and cytosolic phospholipase A(2) (cPLA(2)) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA(2) and cPLA(2) align or are similar to ExoU sequences. Site-specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.  相似文献   

9.
The RNA N-glycosidase activity of ricin A-chain has been characterized. When rat liver ribosomes were used as substrates, the A-chain cleaved the N-glycosidic bond at A-4324 in 28S rRNA. An apparent Michaelis constant (Km) for the reaction was determined to be 2.6 microM and the turnover number (Kcat) was 1777 min-1. When naked rRNA was the substrate, the A-chain cleaved the same bond in 28S rRNA but at a greatly reduced rate. The Km value was 5.8 microM. The results suggest that the A-chain has a similar affinity for 28S rRNA in both ribosomes and the naked states. When the deproteinized Escherichia coli rRNA was the substrates, ricin A-chain cleaved a N-glycosidic bond at A-2600 in 23S rRNA which corresponds to the ricin-site in 28S rRNA of rat liver ribosomes, while the A-chain has little activity on 23S rRNA in the ribosomes. The results suggest that ricin A-chain acts directly on RNA by recognizing a certain structure in the molecules. Using the secondary structure models for each species of rRNA, we have deduced a loop and stem structure having GAGA in the loop to be a minimum requirement for the substrate of ricin A-chain.  相似文献   

10.
11.
The structure and action of ricin, a cytotoxic N-glycosidase   总被引:3,自引:0,他引:3  
The X-ray structure of the heterodimeric plant cytotoxin ricin has been elucidated. The A chain, known to be a specific N-glycosidase, has a prominent active site cleft. The B chain is a two domain lectin, which arose from the replication of a primitive sugar binding peptide. The molecular model reveals residues which are likely to be important in the action of the protein. Site directed mutagenesis reveals the relative importance of a number of these groups. A model for the mechanism of the A chain, including steric strain of the scissile bond and carboxonium ion transition state stabilization is proposed.  相似文献   

12.
13.
Upon jasmonate treatment barley leaf segments express a putative ribosome-inactivating protein (JIP60). The influence of this protein on translation in planta has been analysed by using barley plants and tobacco plants transformed with a barley cDNA encoding JIP60. In both plant systems JIP60 exhibited N-glycosidase activity in vivo. The depurination of the 25S rRNA of tobacco and barley ribosomes led to accumulation of translationally inactive polysomes.  相似文献   

14.
Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity. In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP. When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP. Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect. In contrast, on the protein synthesis of E. coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect. These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E. coli ribosomes. These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity. When the R171K gene was expressed in E. coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected. This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues.  相似文献   

15.
16.
17.
A new group of radioprotective agents, dimethylaminodithiazines, was discovered. The agents are effective when used in relatively small amounts and exert a radioprotective effect rapidly with both intraperitoneal and per os injections. The role of inhibition of oxidative processes and DNA biosynthesis in the mechanism of radioprotective action of dimethylaminodithiazines is discussed.  相似文献   

18.
The effect of citrinin poisoning on rabbit kidney alkaline phosphatase was investigated. After seven days administration of citrinin (2 mg/kg body weight daily) the animals were sacrificed and the level of enzymes estimated in serum and kidney. Serum enzymes showed no variation in activity in the citrinin-treated animals, but in kidney, alkaline phosphatase activity decreased significantly. The decreased activity was mainly associated with the cytoplasmic fraction and in fractions Ib and II. The enzyme II obtained from citrinin-treated animal showed no kinetic difference in substrate specificity, inhibition by phenylalanine, phosphate, sodium-EDTA and Zn2+ ions, activation by Mg2+ ions, thermal inactivation and electrophoretic mobility to that of control Enzyme II. Immunological studies showed that the decrease in enzyme activity was due to existence of inactive enzyme protein. Hormones like cyclic AMP, prostaglandin E1 and parathyroid hormone reversed the decreased enzyme activity due to citrinin poisoning in mouse and rabbit. This study favours the possible existence of active and inactive forms of alkaline phosphatase in the system.  相似文献   

19.
Trichosanthin, a protein from the Chinese medicinal herb Trichosanthes kirilowii, was purified in two essentially quantitative steps involving CM-Sephadex chromatography and reverse-phase high performance liquid chromatography. The protein was found to have a molecular mass of 25-26 kDa, to contain no cysteine, and to contain no glycosidic linkages. Pure trichosanthin was found to have potent abortifacient activity in pregnant mice. In order to understand the molecular basis of this unique biological activity, we have examined the amino acid sequence of the protein. As purified, trichosanthin was found to contain two amino-terminal sequences which differed only in the absence or presence of a tyrosine at residue 1. Sequence analysis of trichosanthin has allowed for determination of the NH2-terminal 38-amino acid residues. Comparison of this sequence to those present in a data base revealed homology with the ricin A-chain. Consistent with this structural homology, we have found that trichosanthin is a potent inhibitor of protein synthesis in a reticulocyte lysate system.  相似文献   

20.
Flavobacterium meningosepticum peptide:N-glycosidase-mediated deglycosylation of N-linked glycan strands of glycoproteins has been found to be strongly influenced by the ionic strength of the assay medium. By use of a modification of a previously published assay procedure for quantitative analysis of glycan release we have been able to improve reproducibility and thus to compare the extent of deglycosylation achieved under a variety of conditions of ionic strength. We have observed that enzyme activity is adversely affected by high ionic strength buffers such as those recommended for deglycosylation of various glycoproteins and recommend the use of low ionic strength buffers for routine use.  相似文献   

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